1. Field of the Invention
The present invention relates to a nucleotide primer set and a detection probe for detecting a genotype of a single-nucleotide polymorphism in an N-acetyltransferase 2 (NAT2) gene.
2. Description of the Related Art
N-acetyltransferase 2 (NAT2) is involved in the metabolism of clinically important pharmaceuticals such as tuberculosis medicine isoniazid (INH) and salazosulfapyridine. Salazosulfapyridine is used for treatment of ulcerative colitis, rheumatoid arthritis, and the like.
It is known that there are genetic polymorphisms in the gene coding NAT2. The phenotype higher in NAT2 activity is called a rapid acetylator (RA), and that lower in activity is called a slow acetylator (SA). It is said that there are four kinds of polymorphisms (NAT2*4, NAT2*5, NAT*6, NAT2*7) in Japanese. NAT2*4 is a wild-type polymorphism. Mutant alleles NAT2*5, NAT*6, and NAT2*7 can be identified by analyzing the genotype of single-nucleotide polymorphisms T341C, G590A, and G857A, respectively. A person having a homozygote or composite heterozygote of the mutant allele is highly likely an SA, and has higher incidence of adverse drug action.
For this reason, identifying the mutation of the NAT2 gene is helpful to avoid adverse drug reactions. It is also possible to select pharmaceutical administration and treatment suitable for individual patient by determining the genotype of the NAT2 gene.
The single-nucleotide polymorphism is generally detected by amplifying a target nucleotide with the Polymerase chain reaction (PCR) method and detecting wild-type and variant amplification products with a specific probe (see, the reference “Jain K. K., Application of Amplicip. CYP450, Mol Diagn. 9, 119-27 (2005)”). However, the PCR method has disadvantages such as complicated procedure of pretreatment including nucleotide extraction, demand for a complex temperature-regulating device such as a thermal cycler, and a longer reaction period of two hours or more. Amplification products by the PCR method are double-stranded chains, and thus, there is a problem in that the complementary chain degrades the detection sensitivity, while functioning as a competitor to the probe during detection. Various methods of converting the amplification product into a single-stranded chain, for example, by decomposing or separating a complementary chain by using an enzyme or magnetic beads, have been studied, but these methods also have a problem in that the operation is complicated and expensive.